Signal-Converting-Proteins (SCP) which are currently known in the art are bi-functional fusion proteins that link an extracellular portion of a type I membrane protein (extracellular amino-terminus), to an extracellular portion of a type II membrane protein (extracellular carboxyl-terminus), forming a fusion protein with two active sides (see for example U.S. Pat. No. 7,569,663). CTLA4-FasL is a SCP in which the N-terminal side is composed of the extracellular domain of CTLA-4, a Type-I membrane protein that binds with high affinity to B7 receptors, and the C-terminal side is composed of extracellular domain of Fas-ligand (FasL), a Type-II membrane ligand that induces cell apoptosis.
CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4), also known as CD152, is a protein receptor that naturally down-regulates the immune system via inhibition of T-cell activation. T-cell activation requires co-stimulatory binding between the CD28 receptor to the CD80 and CD86 receptors, also called B7-1 and B7-2 respectively, on antigen-presenting cells. CTLA-4 is similar in function to CD28 in that they both bind to B7, however, whereas CD28 transmits a positive T-cell activation stimulatory signal, CTLA-4 binds B7 receptors at higher affinity but does not activate T-cells, thus, competing with CD28 and down-regulating the immune-system. The membrane-bound CTLA-4 is known to function as a homodimer that is interconnected by a disulfide bond.
CTLA4's strong binding affinity to B7 led to the design of protein-based therapeutics, linking the CTLA4 extracellular domain to an antibody Fc domain (CTLA4-Fc),that is already approved for use in autoimmune diseases and transplantation (Herrero-Beaumont G, Martinez Calatrava M J, Castañeda S. Abatacept mechanism of action: concordance with its clinical profile. Rheumatol Clin. 2012 March-April 2012; 8(2):78-83). In these chimeric constructs, both the CTLA4 and the Fc domains form a natural homo-dimer (Arora S, Tangirala B, Osadchuk L, Sureshkumar K K. Belatacept: a new biological agent for maintenance of immunosuppression in kidney transplantation. Expert Opinion on Biological Therapy. 2012; 12(7):965-979).
FasL is a Type-II membrane protein that naturally binds and activates Fas-receptors (FasR), which induce cellular apoptosis, or programmed cell death. FasL and FasR belongs to the tumor necrosis factor (TNF) family and FasL/FasR interactions play an important role in the regulation of the immune system and the progression of cancer. FasL, like other TNF super-family members, functions as a non-covalently bound homo-trimeric protein that signals through trimerization of FasR, which usually leads to apoptosis of the “target” cell. Upon FasL binding and trimerization of FasR, a death-inducing signaling complex (DISC) is formed within the target cell, and subsequently apoptosis is induced. Studies indicate that two adjacent trimeric FasL are required for efficient FasR signaling and the formation of DISC (Holler et-al, Molecular and Cellular Biology, February 2003, p. 1428-1440. Eisele et-al, Neuro-Oncology 13(2):155-164, 2011).
Non-Hodgkin lymphomas (NHLs), as a disease set, is among the ten most prevalent malignant tumors, accounting for approximately 4% of all malignancies in both men and women. NHLs are of B or T-lymphocytes lineage with most (80-90%) of them being of B-cell origin. Though prognosis and treatment depend on specific type and stage, irradiation and chemotherapy have been proven effective in many NHL patients. New protein-based therapeutics, such as anti-CD20, have been recently added to the treatment toolbox. The overall 5-year survival rate has increased to approximately 50%, but there is still need for new effective treatment for the more aggressive and relapsing forms of the disease.
Activated B-cells are known to express high levels of B7 receptors, also known as CD80 (B7.1) and CD86 (B7.2), which are required for T-cell activation as part of a co-stimulatory signal between the T-cell CD28 receptor and the B7 receptors on antigen-presenting cells including B lymphocytes. Similarly to activated B-cells, B-cell lymphoma cells also express high levels of B7 molecules.
Signal-Converting-Proteins (SCP) are a novel type of bi-functional fusion proteins that are formed by directly linking an extracellular domain of a type I membrane protein (extracellular amino-terminus), to the extracellular domain of a type II membrane protein (extracellular carboxyl-terminus), creating a fusion protein with two active sides. CTLA4-FasL is one such SCP, in which the N-terminal side is the extracellular domain of CTLA-4 and the C-terminal side is composed of the extracellular domain of Fas-ligand (FasL) (J.H. H, M.L. T. CTLA-4-Fas ligand functions as a trans signal converter protein in bridging antigen-presenting cells and T cells. International Immunology. 2001; 13 (4):529-539). Since CTLA4-FasL has the ability to bind to B7 molecules and to FasR, and in doing so, concurrently, to inhibit co-stimulation and induce apoptosis. CTLA4-FasL has been shown to efficiently induce apoptosis of activated T-cells (Orbach A, Rachmilewitz J, Parnas M, Huang J H, Tykocinski M L, Dranitzki-Elhalel M. CTLA-4. FasL induces early apoptosis of activated T cells by interfering with anti-apoptotic signals. J Immunol. December 2007; 179(11):7287-7294) and to function as a strong immunomodulator in multiple autoimmune and transplantation animal models (Zhang W, Wang F, Wang B, Zhang J, Yu J Y. Intraarticular gene delivery of CTLA4-FasL suppresses experimental arthritis. Int Immunol. June 2012; 24(6):379-388; Jin Y, Qu A, Wang G M, Hao J, Gao X, Xie S. Simultaneous stimulation of Fas-mediated apoptosis and blockade of costimulation prevent autoimmune diabetes in mice induced by multiple low-dose streptozotocin. Gene Ther. June 2004; 11(12):982-991; Shi W, Chen M, Xie L. Prolongation of corneal allograft survival by CTLA4-FasL in a murine model. Graefes Arch Clin Exp Ophthalmol. November 2007; 245(11):1691-1697; Feng Y G, Jin Y Z, Zhang Q Y, Hao J, Wang G M, Xie S S. CTLA4-Fas ligand gene transfer mediated by adenovirus induce long-time survival of murine cardiac allografts. Transplant Proc. June 2005; 37(5):2379-2381). Recently, the present inventors have shown that CTLA4-FasL can induce robust apoptosis of B cell lymphoma cell lines by activating pro-apoptotic signals in parallel to abrogating anti-apoptotic ones (Orbach A, Rachmilewitz J, Shani N, et al. CD40⋅FasL and CTLA-4⋅FasL fusion proteins induce apoptosis in malignant cell lines by dual signaling. Am J Pathol. December 2010; 177(6):3159-3168).